Human stem cell recombineering using viral proteins to catalyze homologous recombination could transform human genome engineering. Bacterial recombineering catalyzes highly efficient in vivo homologous recombination by using a complex of two bacteriophage proteins: one protein is an exonuclease that processes substrate DNA to create single-stranded DNA (ssDNA) ends; the second protein binds to the ssDNA to form a protein-DNA filament that catalyzes homologous DNA pairing and strand exchange. The potential of using bacteriophage recombinases to catalyze human stem cell recombineering is limited by its low efficiency. A possible explanation for low human recombineering efficiency is that viral proteins must coordinate with cellular proteins to catalyze homologous recombination. For instance, the viral DNA binding protein protects the ssDNA recombination substrate from degradation by host nucleases. Furthermore, viral recombineering activity is coordinated with host DNA replication. However, as cellular protein sequences evolve, viral protein sequences must co-evolve to maintain host/viral protein interactions including those required for efficient viral recombination. Recent observations supporting the hypothesis that viral recombineering functions are host-specific include that the phage Che9c recombinase is highly efficient in its host bacterium (M. tuberculosis) but not in E. coli and the E. coli phage A recombinase is not efficient (10- 6) in mouse ES cells but highly efficient (10-1) in E. coli. The hypothesis of this proposal is that recombineering is host-specific and that viral recombinases co-evolved with host proteins. To test this hypothesis I will study the interaction of human and phage recombinases with human and bacterial host proteins and will reconstitute both viral recombinases in human cells and bacterial cells to evaluate the efficiency of recombination. Specific protein-protein and functional interactions will be tested using pull-down assays and enzymatic analysis. The efficiency of recombination will be tested by targeting oligonucleotides to a CFP gene in cells expressing viral recombinases and quantifying the change of CFP to GFP by cell sorting. My long term goals are the improvement of gene targeting specificity and efficiency in human stem cells and the development of human monogenic stem cell disease models. Recombination in human cells with high efficiency and specificity will transform science and medicine. With sharper tools, genome engineering will realize its potential to cure genetic diseases, create human disease models, and achieve the promises of the biotechnology revolution.